Note: Descriptions are shown in the official language in which they were submitted.
-1-
GLP-1 ANALOGS USEFUL FOR DIABETES TREATMENT
Technical Field
The invention relates to the field of improved
pharmaceutical compositions. Specifically, the invention
concerns analogs of the glucagon-like peptide I fragment
7-36 or 7-37 with improved~pharmacological properties.
Background Art
Glucose metabolism is regulated by a number of
peptide hormones, including insulin, glucagon, and
gastric inhibitory peptide (GIP). The complex mechanism
by which these peptide hormones regulate this metabolism
and the manner in which they affect each other is at
least partially elucidated. For example, glucagon binds
to receptors on the surface of the pancreatic beta cells
which produce insulin, and stimulates insulin secretion.
Glucagon-like peptide I has been suggested to stimulate
insulin secretion but this has not been confirmed.
Several of these hormones originate from a mam-
malian glucagon precursor "proglucagon" which is a 180
amino acid peptide. Proteolysis and processing of this
peptide results in a number of these protein hormones;
the results of the processing depend on the origin of the
cells in which this occurs. For example, in the pig and
rat pancreas, proglucagon is processed to form glucagon
and glicentin-related pancreatic peptide, a large peptide
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4~.
which contains both GLP-1 and GLP-2 sequences. In
porcine small intestine, the secreted products are the 69
amino acid glucagon-containing peptide glicentin and the
two glucagon-like sequences;,GLP-1 and GLP-2 as separate
peptides.
In any event, however, the overall sequence of
proglucagon contains the 29 amino acid sequence of
glucagon, the 36 or 37 amino acid sequence of GLP-1 and
the 34 amino acid sequence of GLP-2, separated by amino
acid spacer sequences.
Early attempts to assign a pattern of activity
to GLP-1 gave ambiguous results, and it was subsequently
concluded that truncated forms of this peptide are bio-
logically active. Mojsov, S., et al. J Clin Invest
(1987) 79:616-619 disclose that only the 3l amino acid
peptide GLP-1 (7-37) strongly stimulates the release of
insulin from pancreas;.although both the truncated and
full length 37 amino acid form had earlier been found in
pancreas and intestine: It has been demonstrated that
GLP-1 (7-36), possibly with the carboxy terminus
amidated, is also a potent mediator of insulin release.
(See, e.g., ~Iolst, J.J., et al. ~EBS,~etters (1987)
,~,',~,: 169-174 ) .
The invention described below concerns analogs
of these truncated forms of GLP-1, which. have desirable
combinations of characteristics as they relate to potency
in potentiating glucose-induced insulin secretion'and
glucose-induced inhibition of glucagon secretion and to
circulating half-life. The physiological effects of the
truncated forms in potentiating glucose-induced insulin
secretion have been shown as described above by Holst,
J.J., et al. and Mojsov, S., et al. (supra). The
activity of the truncated hormones in inhibiting glucagon
release,has been shown by Orskov, C., et al. ~Endocrinol
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(1988) 123:2009-2013; Suzuki, S., et al. Diabetes
Research: Clinical Practice (1988) 5_(Supp. 1):530. The
circulating half-life of these truncated forms is
short--approximately four minutes as shown by Kreymann et
al. The Lancet (December 5, 1987) 1300-1303. The
modified forms of these truncated GLP-1 peptides provide
the opportunity to optimize these properties.
There is some literature relating to the study
of degradation of peptide hormones in the liver and in
plasma and the half-life of such hormones in vivo
generally. An early paper by McDonald, J.'K. et al., s
Biol Chem (1969) 244:6199-6208 showed that a dipeptidase
was responsible for the degradation of glucagon in rat
liver. Studies on the growth hormone releasing factor, a
member of the general glucagon, GLP-l, GLP-2 family, was
shown to be rapidly degraded in plasma ~ vitro and also
,~,n y yo by a dipeptidase, (Frohman, L.A. et al., C 'n
Invest (1986) ~g:9b6-913). Murphy, W.A. et al:, in
Pe~~tide Research (1988) ,i:36-41, showed that some but not
all alkylated growth hormone releasing factor peptides
had higher potency "~ vivo. In particular, for example,
the triisopropylated GRF-29 was found to be 106 times
more active'than GRF-29 itself. On the other hand, 6RF-
29 which was in methylated at the N-terminus was only 40%
as potent as the parent. Itvwas also shown that
substitution of D-Ala position 2 of this hormone enhanced
its potency. It was, of course, not certain to what
effect on properties the enhancement of potency could be
attributed.
Others have attempted some modifications of
GLP-1 (7-37). It has been shown that deletion of the
histidine residue at position 7 greatly diminishes the
activity of the hormone (Suzuki, S., et al. (supra);
Hendrick, G.K., et al. Abstract: Endocrine Society
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WO 91!11457 PCT/US91/00500
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r.~'~'.
Meeting, New Orleans LA (1988)). There have been
conflicting reports concerning the effect of one or more
C-terminal deletions (Suzuki, S., et al. (supra);
Yanaihara, C., et al. Abstract for A Glucacron and Related
Pertides Satellite Symposium, 8th International Congress
of Endocrinology, July 15-16, 1988, Osaka, Japan).
However, there is an extensive literature with regard to
modifications of other members of this peptide hormone
family, such as GIP, glucagon releasing factor (GRF),
secretin and vasoactive intestinal peptide (VIP).
pisclosure of the Invention
The invention provides modified forms of the
GLP-1 (7-34);,(7-35); (7-36) or (7-37) human peptide or
the C-terminal amidated forms thereof. The native
peptides have the amino acid sequence:
7 10 15 20 25
H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-
30 37
K-E-F-I-A-iV-L-V-K-(G)-(R)-(G)
wherein (G), (R), and (G) are present or absent depending
on indicated chain length. The modified forms contain
one or more alterations of the native structure and are
of improved ability for therapeutic use. Either the
modified forms have greater potency than glucagon to.
potentiate insulin secretion or enhanced stability in
plasma or both. This potency and enhanced stability can
be assessed as described below.
The standard one letter abbreviation code for amino
acids is used.
The analogs of the invention which show enhanced
insulin stimulating properties have the foregoing
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sequence, or the C-terminal amide thereof, with at least
one modification selected from the group consisting of:
(a) substitution of a neutral amino acid, arginine,
or a D form of lysine for lysine at position 26 and/or 34
and/or a neutral amino acid, lysine, or a D form of
arginine for arginine at position 36;
(b) substitution of an oxidation-resistant amino
acid for tryptophan at position~3l;
(c) substitution according to ut least one of:
Y~for V at position 16;
K for S at position 18;
D for E at position 21;
S for G at position 22;
R for Q at position 23;
R for A at position 24; and
Q for K at position 26;
(d) a substitution comprising at least one of:
an alternative small neutral amino acid for A
at position'8;
an alternative acidic amino acid or neutral
amino acid for E at position 9;
an alternative neutral amino acid for G at
position l0; and
an alternative acidic amino acid for.D at
position 15; and
(e) substitution of an alternative neutral amino
acid or the D or N-acylated or alkylated form of
histidine for histidine at position Z.
With respect to modifications (a), (b), (d) and.
(e), the substituted amino acids may be in the D form; as
indicated by a superscript t, e.g., C~. The amino acids
substituted at position 7 can also be in the N-acylated
or N-alkylated forms.
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Thus, one aspect of the invention is directed to
peptides having enhanced insulin stimulating properties
analogous to the above-mentioned truncated forms of GLP-1
(7-34) to GLP-1 (7-37), as described above.
In another aspect, the invention is directed to
peptides which show enhanced degradation resistance in
plasma as compared to GLP-1 (7-37) wherein this enhanced
resistance to degradation is defined as set forth below.
In these analogs, any of the above-mentioned truncated
forms of GLP-1 (7-34) to GLP-1 (7-37) or their C-terminal
amidated forms is modified by
(a) substitution of a D-neutral or D-acidic amino
acid for H at position 7, or
(b) substitution of a D-amino acid for A at
position 8, or
(c) both, or
(d) substitution of an N-acylated or N-alkylated
form of any naturally occurring amino acid for H at
position 7.
Thus, analogs of the invention which are resistant
to degradation include (N-acyl (1-6C) AA)7 GLP-1 (7-37)
and (N-alkyl (1-6C) AA)7 GLP-1 (7-37) wherein when AA is
a lysyl residue, one or both nitrogens may be alkylated
or acylated. AA symbolizes any amino acid consistent
with retention of insulin stimulating activity.
For substitutions of D-amino acids in the 7 and 8
positions, the D residue of any acidic or neutral amino
acid can be used at position 7 and of any amino acid at
position 8, again consistent with insulin stimulating
activity. Either or both of position 7 and 8 can be
substituted by a D-amino acid; the D-amino acid at
position 7 can also be acylated or alkylated as set forth
above. These modified forms are applicable not only to
GLP-1 (7-37) but also the shorter truncated analogs as set forth above.
According to a first aspect of the invention, there is provided a
peptide which is more potent than glucagon in stimulating insulin release from
islet cells, said peptide consisting of labeled or unlabeled GLP-1 (7-34), GLP-
1 (7-
35), GLP-1 (7-36), or GLP-1 (7-37) or the C-terminal amide form thereof,
having at
least one modification selected from the group consisting of: (a) substitution
of a
neutral amino acid, arginine, or a D form of lysine for lysine at position 26
and/or
34 and/or a neutral amino acid, lysine or a D form of arginine for arginine at
position 36; (b) substitution of an oxidation-resistant amino acid for
tryptophan at
position 31; (c) substitution according to at least one of: Y for V at
position 16; K
for S at position 18; D for E at position 21; S for G at position 22; R for Q
at
position 23; R for A at position 24; and Q for K at position 26; (d) a
substitution
consisting of at least one of: an alternative small neutral amino acid for A
at
position 8; an alternative acidic amino acid or neutral amino acid for E at
position
9; an alternative neutral amino acid for G at position 10; and an alternative
acidic
amino acid for D at position 15; and (e) substitution of an alternative
neutral amino
acid or the D or N-acylated or alkylated form of histidine for histidine at
position 7,
wherein for (a), (b), (d) and (e), the substituted amino acids can optionally
be in
the D form and the amino acids substituted at position 7 can optionally be in
the
N-acylated or N-alkylated form.
According to a second aspect of the invention, there is provided a
peptide useful to release insulin from islet cells, said peptide having
enhanced
resistance to degradation in plasma as compared to GLP-1 (7-37) and said
peptide consisting of labeled or unlabeled GLP-1 (7-34), GLP-1 (7-35), GLP-1
(7
a
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-36), or GLP-1 (7-37) or the C-terminal amide form thereof, having at least
one
modification selected from the group consisting of: (a) substitution of the D
form of
a neutral or acidic amino acid or the D form of histidine for histidine at
position7;
(b) substitution of a D form of an amino acid for alanine at position 8; and
(c)
substitution of an N-acylated (1-6C) or N-alkylated (1-6C) form of an
alternate
amino acid or of histidine for histidine at position 7..
According to a third aspect of the invention, there is provided a
pharmaceutical composition useful in the treatment of diabetes Type II which
comprises an effective amount of the peptide described above in admixture with
a
pharmaceutically acceptable excipient.
According to a fourth aspect of the invention, there is provided a
peptide which is more potent than glucagon in stimulating insulin release from
islet cells, and said peptide consisting of labeled or unlabeled analogs of
truncated forms of GLP-1, which consist of GLP-1 (7-34), GLP-1 (7-35), GLP-1
(7-
36), or GLP-1 (7-37) or the C-terminal amide form thereof, wherein the analog
is
selected from the group consisting of: (Ht)'-GLP-1 (7-37), (Y)'-GLP-1 (7-37),
(N-
acetyl-H)'-GLP-1 (7-37), (N-isopropyl-H)'-GLP-1 (7-37), (At)$-GLP-1 (7-37),
(Et)s-
GLP-1 (7-37), (D)s-GLP-1 (7-37), (Dt)s-GLP-1 (7-37),(Ft)'°-GLP-1
(7-37),
(S)ZZ(R)ZS(R)Za(Q)zs-GLP-1 (7-37), and (S)$(Q)s(Y)'6(K)'8(D)Z'-GLP-1 (7-37).
In other aspects, the invention is directed to pharmaceutical
compositions containing one or more of these peptides as active ingredients
and
to methods to treat Type II diabetes using these peptides or compositions
thereof.
v
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Brief Description of the Drawings
Figure 1 schematically outlines the classification of amino acids as
used herein.
Figure 2 gives a list of various compounds of the invention.
Figure 3 shows the results of radiolabel sequencing analysis for
degradation of two analogs in plasma.
Figure 4 shows the results of various GLP-1 (7-37) analogs with
changes in the amino terminal region, to displace 'zsl-GLP-1 (7-39) from amino
terminal specific antiserum.
Modes of Carryinp Out the Invention
The analogs of the invention, which are modified forms of the GLP-1
(7-34), (7-35), (7-36) or (7-37) are characterized by showing greater potency
than
glucagon in an in vitro assay measuring insulin release from isolated rat
islets in
culture, or by enhanced stability in plasma or both.
Assays for Analogs with Enhanced Insulin Release Stimulating
Properties
One group of analogs of the invention is more potent than glucagon
in stimulating insulin release from islet cells. By being "more potent than
glucagon
in stimulating insulin release from islet cells" is meant that the analog
referred to
shows greater potency in an
CA 02073856 2000-02-22
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in vitro assay selected from the group consisting of the
following: Rat islets for these assays are isolated by
the method of Sutton, R. et al., Transplantation (1986)
42:689-691.. _ Briefly,
Sprague-Dawley male rats are anesthetized and the lower
end of the common bile duct is cannulated with a 2 FG
cannula tied in place. The left and right hepatic ducts
are then ligated separately above the region of the entry
of pancreatic ducts into the biliary tree. The rats are
killed by exsanguination and 3 mL Hank's solution
containing 7.5 mM CaCl2, 20 mM HEPES buffer and 1-6 mg/mL
Type I collagenase are run into the cannula to uniformly
distend the pancreas. The pancreas is then excised and
placed in a beaker on ice prior to incubation in Hank's
solution containing 20 mM HEPES buffer at 37°C.
After 13-25 min of incubation, the pancreas is
removed and placed in Hank's solution containing 5 g/1
bovine serum albumin and 20 mM HEPES buffer at 4°C. All
of the pancreatic tissue is then gently syringed through
a 14 FG needle, suspended in further Hank's solution
containing HEPES as above, centrifuged at 50 g for 10 sec
and the supernatant is discarded. The tissue pellet is
resuspended and again gently syringed, followed by
another wash, after which the dispersed tissue is passed
through a nylon mesh filter of 500 a pore size. The
filtered tissue is centrifuged at 350 g for 5 sec, the
supernatant discarded, and the tissue is then suspended
in 25% Ficoll made up in Hank's with HEPES as above, on
which was layered a discontinuous density gradient of
23%, 20%, and 1l% Ficoll solutions. This density
gradient was spun at 750 g for 10 min at 4°C, and the
tissue obtained from the upper two interfaces was washed
three times in Hank's solution and viewed through a
dissecting microscope for hand kicking of islets.
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In one approach the ability of the GLP-1 analog to
potentiate secretion from these Islets is then determined
according to the method of Schatz, H. et al., in "Methods
in Diabetes Research" (1984)- Volume 1, Part C: pages
291-307, In this
method, 5-10 islets per test tube are incubated in 1 mL
Krebs-Ringer-bicarbonate buffer (KRB buffer). For
testing, glucagon or the modified analog of the invention
is added at 5-10 ~cg/mL. The level of insulin released
may be measured by the method of Jensen, S.L. et al., M J
Phvsiol (1978) 235:E381-E386;
The following protocol is a preferred method to
measure stimulation of insulin secretion. After
collagenase digestion, the islets are allowed to recover
overnight by incubation in DMEM (.Dulbecco's Modified
Eagle Medium 16 w/o glucose), 2.8 mM glucose, 10% fetal
bovine serum (FBS) at 37oC, 5% C02.
The next day, islets to be used for the experiment
are transferred to DMEM, no glucose, 0.2% BSA (Armour,
clinical grade, made at 5% stock) for a 60 min
preincubation in serum-free, glucose-free medium. Islets
are picked up by Eppendorf*pipette and transferred to 60
mm TC plates containing 8.0 mL medium and returned to the
incubator for 60 min. Islets are counted, during this
transfer. (Note: each data point is 5 islets,
experiments are usually performed in quadruplicate;
therefore, 20 islets are used per data point.)
Typically, recoveries are 150-200 islets per pancreas.
Any suspect islets--too ragged or falling apart--are not
used.
During the 60 min preincubation, the experiment is
set up, so that all that is needed at the end of the
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WO 91 / 11457 ' PCT/US 91 f 00500
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preincubation is to transfer islets in groups of 5 to
experimental conditions. The experiment is set up in 48
well TC plates with 0.5 mL medium per well. To DMEM-0.2o
BSA is added glucose to desired concentration (usually
2.8 mM for hypoglycemic conditions, 5.6 mM glucose for
euglycemic, or 16.7 mM glucose for hyperglycemic) and
test compound at various dose ranges (typically, 1 pM to
100 nM). Test compound is diluted from stock stored at
-80°C and at -0.3 mM serially into phosphate buffered
saline (PBS) 0.2% BSA to prevent loss on sides of tubes.
After medium plus test compound is mixed,~0.5 mL each is
added to 4 wells for quadruplicate data points.
After the preincubation period, 5 islets are added
per well. Islets are picked up by eppendorf pipette in
25 u1 volume. Incubation continues another 60 min; at
which time 0.3 mL is harvested per well with care taken
not to pick up islets. Wells are then rechecked for
islet number. Medium is then assayed for insulin content
using an insulin RIA. If medium is not immediately
assayed, it is stored at -20°C until assay. Dose
response curves for insulin secretion are plotted and
ED50 is calculated from the curves.
Higher potency as compared to glucagon is defined
as either higher levels of insulin released by the analog
using the same concentrations of glucagon and analog or,
alternatively, the same level of insulin release but
using a lower concentration of analog than glucagon.
While the foregoing assays form specific criteria
for judging enhanced potency, alternative assays can also
be used as substitutes for those set forth above.
An additional test for potency of the compounds of
the invention.measures their ability to stimulate cAMP
production in RIN 1046-38 cells. This assay can be
conducted as follows:
..~ 91/11457 ' PCT/US91/00500
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On day 1, 5 x 105 RIN 1046-38 cells (Drucker, D.J.,
et al., Proc Natl Acad Sci USA (1987) 84:3434-3438) are
seeded into individual wells of 6-well dishes with 2.5 mL
M199 culture medium. On day 4, cells are re-fed with
fresh medium and on day 5 the assay is performed. At
this time there are -2.0-2.5 x 106 cells per well.
Assays are only performed on cell passage 524.
At time -60 min, monolayers are washed twice with
2.5 mL PBS, and medium is changed to 1.0 mL of DMEM
medium plus 4.5 g/1 glucose and 0.1% BSA (assay medium).
At 0 time, medium is aspirated and fresh assay medium,
1.0 mL, containing test compound is added. Test compound
is added in 50 u1 volume of PBS plus 0.1% BSA; controls
are added in vehicle alone. Incubation is continued for
0 to 60 min.
At termination, conditioned medium and monolayer
are harvested to measure both extra- and intracellular
cAMP content. For extracellular measurement, medium is
removed and centrifuged to remove any cellular debris.
For intracellular determination, after medium removal,
1.0 mL of ice cold 95% ethanol is added to monolayer.
Cells are collected by scraping, lysed by two.cycles of
quick freeze/thawing using liquid N2, and cell debris
then removed by centrifugation. Aliquots (1/40th well
content) of conditioned medium and ethanol cell extract
are measured in duplicate far CAMP levels using an RIA
kit by the acetylated protocol.
As above, higher potency as compared to glucagon is
defined either as higher cAMP stimulation by both the
arialog and glucagon at the same concentration, or the
sane cAMP stimulation by the analog at a lower
concentration.
Still other assays for measurement of enhanced
' 35 potency to mediate insulin release can be used.
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The ability of the compounds to potentiate the
release of insulin can be tested both in vitro and in
viyo. Insulin released can be detected using a standard
antibody assay both in analyzing plasma in in vivo
studies and in analyzing media or perfusion liquid in
vitro.
For example, a useful in vitro assay uses the
pancreatic infusion assay method of Penhos, J.C., et al.
_D~;~:betes (1969) 18:733-738, as employed in the method of
Weir, G.C., et al. J Clin Investiqat (1974) 54:1403-1412.
Insulin secretion can also be measured by the method
described by Holst, J.J., et al. FEBS Letters (1987)
21:169-174 (supra). Also useful as an assay for
insulinotropic effect is the measurement of stimulation
of adenylate cyclase in the RIN 1046-38 cell line.
Drucker, D.J. et al., Proc Natl Acad Sci USA (1987)
$,8,:3434-3438 (supra).
Inhibition of glucagon release can be shown as
described by Orstov, C., et al. Endocrinol (1988)
,~,,~"~,:2009-2013; Suzuki, S. , et al. Diabetes Research:
Clinical Practice (1988) ~(Supp. 1):530 (both supra).
Ae~a~s for Enhanced Stabilitv to Degradation
The therapeutic efficiency of the GLP-1 analogs of
the invention can also be enhanced by providing analogs
with increased half-lives in vivo. By "enhanced half-
life ',fin vivo" is meant a demonstrated ability to resist
degradation in the presence of plasma according to an
assay selected from the group consisting of the
following. In all assays, the plasma is prepared by
collecting blood into heparinized tubes, placing the
tubes on ice and centrifuging at about 3,000 rpm for l0
minutes in a tabletop centrifuge. The separated plasma
is stored at 4°C.
r.
-13-
A. Radiolabel Sequencing:
The GLP analog is labeled by radio-iodination in
position 19 using standard radiolabeling methods. After
exchange into RIA buffer (5~ mM NaHP04 pH 7.4, 0.25% BSA
(Armour insulin and FFA free), 0.5% BME, 0.002%
polylysine (Sigma 15,000 mw), 0.05% Tween 20, 0.1% NaN3),
the radioiodinated peptide (about 105 cpm/50 mL) and cold
uniodinated peptide (20 ~1 100 nM) are added into 2 ml of
plasma to a final concentration of 1 nM and incubated in
l0 a circulating water bath for preset times. Total RIA
buffer added to plasma never exceeds 5% of total volume.
At the end of incubation, 10% bacitracin (w/v) in water
is added to a final concentration of 0.1% to stop the
reaction.
The plasma is then extracted using C18 Sep-Pak to
separate the analog and any fragments from the bulk of
the plasma proteins. Sep-Pak cartridges (Waters)* are
washed with 2 mL of 1-propanol, followed by 2 mL of water
and then equilibrated with 2 mL of 20% CH3CN containing
0.1% trifluoroacetic acid (TFA) (Buffer A).
The bacitracin-treated plasma is made 20% CH3CN
with CH3CN containing 0.1% TFA and is expressed slowly
through a 3 mL plastic syringe through the cartridge.
The cartridge is then washed with two 1 mL Buffer A
washes and eluted with a single 2 mL wash of 50% CH3CN
containing 0.1% TFA (Buffer B) into a siliconized 12 x 75
glass tube. Recovery of the analog or fragments is more
than 90%.
The eluates are concentrated to 100 ~1 in a Speed
vac and transferred to a 1.5 mL Eppendorf tube to which a
1 mL RIA buffer rinse of the original tube had been
added.
To purify any analog or its fragments when the
analogs of GLP-1 (7-37) are used, the concentrates are
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CA 02073856 2000-02-22
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treated with 5 ~1 of antiserum prepared to a synthetic
peptide corresponding to residues 24-37 which recognizes
GLP-1, GLP-1 (7-37) but not GLP-1 (7-36). When the
shorter forms of analogs ark used, alternate carboxy
terminal-specific antisera (prepared in the same manner
but using a peptide corresponding to residues 24-34, 24-
35 or 24-36 as immunogen) are used. To this is added 100
~1 of a 10% (w/v) solution of protein A-Sepharose*
(Pharmacia) in PBS, and the mixture is incubated
overnight at 4°C with gentle rocking. The Sepharose is
then pelleted with a 5 second spin iri an Eppendorf
centrifuge at 4°C after which the pellet is washed two
times with cold RIA buffer and four times with cold PBS.
Polyclonal antisera were raised in New Zealand
White rabbits against a synthetic peptide fragment
corresponding to residues 24 to 37 of GLP-1 (7-37) using
the method of Mosjoy, S. et al.,~J Biol Chem (1986)
x:11880-11889. Initial immunizations were into the
inguinal lymph nodes and used Freund's complete adjuvant.
Two subcutaneous boosts were performed at 1 week
intervals after the initial immunization and used
Freund's incomplete adjuvant. For a single immunization
or boost 100 ~g peptide and 100 ~g methylated BSA
dissolved in 0.3 mL phosphate-buffered saline (PBS) were
emulsified with 0.9 mL adjuvant. Bleeds (50 mL) began at
week 6 after the initial immunization and continued at 1
month intervals thereafter. Repeat boosts were performed
as above when titers dropped noticeably from the level of
the previous bleed.
' Serum was prepared by allowing the blood to clot
overnight at 4°C. The clot was pelleted by
centrifugation at 2000 g for 15 minutes and the serum
removed. Serum is stored in aliquots at -20 or -80°C.
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.~0 91/11457 ' PCT/US91/00500
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The peptides are then eluted from the antibody
protein-A sepharose complex with three 100 ~1 washes of
Buffer B. The combined 300 ~,1 of wash are then applied
directly to an ABI model 477 sequencer used according to
the manufacturer's instructions. Fractions from each
cycle are then diverted for,counting. Counting can be
effected in 4 mL aqueous scintillant (ACS, Amersham).
The cycle at which label appears indicates the
extent of degradation from the N-terminus. If no
degradation from the N-terminus has occurred in the GLP-
1 (7-37) analog, all of the label will appear in the 13th
cycle, corresponding to the tyrosine at position 19; if
degradation has occurred, the label will appear in
earlier cycles.
B. Assay by RP-HPLC:
While the foregoing method is a clear criterion for
exhibiting a longer half-life in plasma, alternative
forms of the assay for this property can also be used.
In one convenient assay, the analog can be assessed for
degradation into fragments using reverse phase-HPLC,
since the fragments have different retention times from
the analog per se: In this assay, the analog is added to
plasma for various times and recovered similarly to the
method described above for radiolabel sequencing
analysis. Specifically, the analog at a concentration of
100 nM in RIA buffer is spiked into 1 mL plasma to a
' final concentration of 1 nM and after incubation in 37°C
circulating water bath for various preset times, the
reaction is stopped by bringing the plasma to 0.1% (w/v)
in bacitracin:
The peptides are then purified by Sep-Pak
extraction as described above. The eluates are
concentrated to about 1 mL on a Speed-vac, diluted with 1
mL distilled water, frozen at 80°C and lyophilized
-16-
overnight. The powder is resuspended in 0.5 mL Buffer C
(0.1% TFA in water) per mL starting plasma and 0.25 mL
are injected on a Hewlett-Packard 1090L liquid
chromatograph using an Allt~h* C18 column (0.45 x 25 cm;
10 ~m particle size) with a Brownlee*2 cm C18 guard
column. The extraction is monitored at OD214 throughout
the run and the solvent flow rate was 1 mL/minute. A
gradient between Buffer C and Buffer D (0.1% TFA in
acetonitrile) is set up over a 40 minute run time. The
gradient starts at 35% D is held for the first 2 minutes
after injection and then increased to 42% D over 24
minutes. The gradient is then increased to 60% D over
the next two minutes, held at this level for 2 minutes
and returned to 35% D over the next 2 minutes. The %D
remains at 35% for the remaining 8 minutes of the run.
Fractions are collected at 0.5 minute intervals for the
first 30 minutes of each run and dried in a Speed-vac.
The samples can be assayed for the presence of analog or
fragment using RIA (measuring competition with labeled
GLP-1 (7-37) for binding to C-terminal specific
antiserum) or by any conventional or convenient
alternative method.
Radioimmunoassays for the amino or carboxyl
terminus of GLP-1 (7-37) use a single antibody
displacement format. Binding of 1251-GLP-1 (7-37) to
antibody is incrementally displaced by increasing
concentrations of unlabeled peptide in solution.
Antibody bound iodinated peptide is separated from free
iodinated peptide in solution by precipitation of the
antibody-peptide complex with Pansorbin"' (Boheringer
Mannheim). The resulting pellet is then counted on a
gamma counter.
*Trademark
7
CA 02073856 2000-02-22
-17-
C. Loss of Binding to N-Terminal Specific
antibodies:
A third approach to assessment of half-life in
plasma utilizes polyclonal ar monoclonal antibodies
specifically prepared to the N-terminus which will fail
to bind degraded analog. These antisera were raised
against a synthetic peptide corresponding to GLP-1 (7-
22) which contains an additional cysteine residue at the
carboxyl terminus and is specifically coupled to KLH via
the cysteine using mal-sac-HSNA as described by Aldwin,
L. et al. Analytical Biochem (1987) 164:484-501.
Polyclonal antibodies were generated in New Zealand white
rabbits by giving a primary immunization into the
inguinal lymph nodes of 500 ~g conjugate emulsified with
Freund's complete adjuvant and then two subsequent boosts
of 200 ~g each in Freund's incomplete adjuvant at 2 week
intervals. Blood (50 mL) is collected monthly thereafter
and boosts are performed if titers are low. For
generation of monoclonal antibodies, Balb/c mice were
immunized intraperitoneally with 200 u8 of conjugate in
0.5 ml Freund's complete adjuvant. Mice were boosted
biweekly with 100 ~g conjugate in 0.5 ml Freund's
incomplete adjuvant. Cells isolated from the spleens of
these mice were fused with Fox-NY cells to produce
monoclonal cell lines. Monoclonal secreting cell lines
are produced using the standard Kohler-Millstein
technology. Monoclonal supernatants and polyclonal sera
are screened using an ELISA method for binding to GLP-1
(7-37) but not to GLP-1 (8-37). The specificity is
confirmed in standard solution phase RIA.
The kinetics of degradation of GLP-1 (7-37) are
followed by adding the analog to human plasma in RIA
buffer, generally 10 ~L of 100 x concentrated peptide is
added to 1 mL of plasma to obtain the desired
*Trademark
CA 02073856 2000-02-22
~~~'la~
T
WO 91/11457 ' PCT/US91/00500
-18-
concentration; the sample is then incubated in 37°C water
bath and triplicate 50 JCL aliquots are removed at various
times.. The aliquots are immediately ethanol precipitated
for radioimmunoassay using a-competition for binding of
the N-terminal specific antibody with radioiodinated GLP-
1 (7-37). Disappearance of the ability to compete with
the radioiodinated GLP-1 (7-37) peptide indicates
degradation of the analog. .
In any of these assays, the analog tested has
enhanced stability if it is less rapidly degraded than
GLP-1 (7-37).
The Analoas
The analogs of the invention having higher potency
than glucagon or having enhanced degradation resistance
are modified forms of GLP-1 (7-34) through GLP-1 (7-37)
wherein, in some instances, amino acids of certain
classes are substituted for the naturally occurring
residues.
Amino acid residues can be generally subclassified
into four major subclasses as follows and as shown in
Figure 1. _
Acidic: The residue has a negative charge due to
loss of H ion at physiological pH and the residue is
attracted by aqueous solution so as to seek the surface
positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium at
physiological pH.
Basic: The residue has a positive charge due to
association with H ion at physiological pH and the
residue is attracted by aqueous solution so as to seek
the surface positions in the conformation of a peptide in
which it is contained when the peptide is in aqueous
medium at physiological pH.
.. V 91/11457 ' PCT/US91/00500
-19-
Neutral/nonpolar: The residues are not charged at
physiological pH and the residue is repelled by aqueous
solution so ws to seek the inner positions in the
conformation of a peptide iir which it is contained when
the peptide is in aqueous medium. These residues are
also designated "hydrophobic" herein.
Neutral/polar: The residues are not charged at
physiological pH, but the residue is attracted by aqueous
solution so as to seek the outer positions in
the conformation of a peptide in which it is contained
when the peptide is in aqueous medium.
It j.s understood, of course, that in a statistical
collection of individual residue molecules some molecules
will be charged, and some not, and there will be an
attraction for or repulsion from an aqueous medium to a
greater or lesser extent. To fit the definition of,
"charged", a significant percentage (at least ap-
proximately 25%).of the individual molecules are charged
at physiological pH. The degree of attraction or repul-
sion required for classification as polar or nonpolar is
arbitrary, and, therefore, amino acids specifically
contemplated by the invention have been specifically
classified as one or the other. Most amino acids not
specifically named can be classified on the basis of
known behavior.
Amino acid residues can be further subclassified as
cyclic or noncyclic, and aromatic or nonaromatic,
self-explanatory classifications with respect to the side
chain substituent groups of the residues, and as small or
large. The residue is considered small if it contains a
total of 4 carbon atoms or less, inclusive of the
carboxyl carbon. Small residues are, of course, always
nonaromatic. '
VI'O 91/11457 ' PCT/US91/00500 ,
-20-
For the naturally occurring protein amino acids,
subclassification according to the foregoing scheme is as
follows (see also Figure 1).
Acidic: Aspartic acid and Glutamic acid;
Basic/noncvclic: Arginine, Lysine;
Basic/cvclic: Histidine;
Neutral/polar/small: Glycine, Serine and
Cysteine;
Neutral/nolar/large/nonaromatic: Threonine,
Asparagine, Glutamine;
Neutra,jpola g~e/aromatic: Tyrosine;
Neutral/nor~polar/sma~i: Alanine
J
Neutral/nonpolar/la,~c~ejnonaromatic: Valine,
Isoleucine, Leucine, Methionine;
Neutral/nonnolar/larae/aromatic: Phenylalanine,
and Tryptophan.
The gene-encoded amino acid proline, although
technically within the group
neutral/nonpolar/large/cyclic and nonaromatic, is a
special case due to its known effects on the secondary
conformation of peptide chains, and is not, therefore,
included in this specific defined group.
Certain commonly encountered amino acids, which are
not encoded by the genetic code, include, for example,
2~ ~ ~~~~~
.v0 91/11457 PCT/US91/00500
-21-
beta-alanine (beta-ala), or other omega-amino acids, such
as 3-amino propionic, 4-amino butyric and so forth,
alpha-aminoisobutyric acid (Aib), sarcosine (Sar),
ornithine (Orn), citrulline -(Cit), homoarginine (Har),
t-butylalanine (t-BUA), t-butylglycine (t-BuG),
N-methylisoleucine (N-MeIle), phenylglycine (Phg), and
cyclohexylalanine (Cha), norleucine (Nle), cysteic acid
(Cya) and methionine sulfoxide (MSO).. These also fall
conveniently into particular categories.
Based on the above definition,
Sar and beta-ala are neutral/nonpolar/small;
t-BuA, t-BuG, N-MeIle, Nle and Cha are
neutral/nonpolar/large/nonaromatic;
Har and Orn are basic/noncyclic;
Cya is acidic;
Cit, Acetyl Lys, and MSO are
neutral/polar/large/nonaromatic; and
Phg is neutral/nonpolar/large/aromatic.
See, also, Figure 1.
The various omega-amino acids are classified
according to site as neutral/nonpolar/small (beta-ala,
i.e., 3-aminopropionic, 4-aminobutyric) or large_(all
others).
Other amino acid substitutions'for those encoded in
the gene can also be included in peptide compounds within
the scope of the invention and can be classified within
this general scheme.
The nomenclature used to describe GLP-l analog
compounds of the present invention follows the
conventional practice wherein the amino group is assumed
to the left and the carboxy group to the right of each
amino acid in the peptide. In the formulas representing
selected specific embodiments of the present invention,
the amino- and carboxy-terminal groups, although often
fVO 9I/11457 ' PCf/US91/00500
-22-
s:,~~.
not specifically shown, will he understood to be in the
form they would assume at physiological pH values, unless
otherwise specified. Thus, the N-terminal H+2 and
C-terminal-O at physiological pH are understood to be
present though not necessarily specified and shown,
either in specific examples or in generic formulas.
The foregoing describes the status of the termini
at neutral pH; it is understood, of course, that the acid
addition salts or the basic salts of the peptides are
also included within the scope of the invention. At high
pH, basic salts of the C-terminus and carboxyl-containing
side chains may be formed from nontoxic pharmaceutically
acceptable bases, and suitable counter- ions include, for
example, Na+, K+, Ca++ and the like. Suitable
pharmaceutically acceptable nontoxic organic cations can
also be used as counter ions. In addition, as set forth
above, the peptides may be prepared as the corresponding
amides.
Sditable acid addition salts with respect to the N-
2o terminus or amino group-containing side chains include
the salts formed from inorganic acids such as
hydrochloric, sulfuric, or phosphoric acid and those
formed from organic acids such as acetic, citric, or
other pharmaceutically acceptable nontoxic acids.
In the peptides shown, each encoded residue where
appropriate is represented by a single letter
designation, corresponding to the trivial name of the
amino acid, in accordance with the following conventional
list:
35
~~~~~ is
.~O 91/11457 PCT/US91/00500
-23-
One-Letter
Amino Acid Symbol
Alanine A
Arginine R
Asparagine N
Aspartic acid D
Cysteine C
Glutamine Q
Glutamic acid E
Glycine G
Histidine H
Isoleucine I
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W
Tyrosine Y
Valine v -
The amino acids not encoded genetically are ab-
breviated as indicated above.
In the specific peptides of the present applica-
tion, the L-form of any amino acid residue having an
optical isomer is intended unless otherwise expressly
indicated by a dagger (t) superscript. While the
residues in the analogs of the invention peptides are
normally in the natural L optical isomer form, one or
two, preferably one, amino acid in addition to a
specified "same-amino-acid-D form," substitution for the
~~'l;ocit~t~
WO 91/11457 ' PCT/US91/00500
-24-
naturally occurring amino acid may be in the D
configuration.
In the notation used in designating specific
analogs, the positions modified are shown as superscripts
to the replacement amino acid; thus, (H'~)7-GLP-1(7-37) is
the noted GLP-1(7-37) form with the D form of histidine
substituted at position 7;
(S)22(R)23(R)24(Q)26-GLP-1(7-37) refers to the 7-37 GLP
form with serine at position 22, arginine at positions 23
and 24, and glutamine at position 26.
Preferred Embodiments
A. Enhanced Stimulatorv Analogs
For analogs with increased insulin-stimulating
activity, particularly preferred analog compositions of
the invention are those wherein only limited numbers of
modifications or substitutions, as compared to GLP-1
truncated forms are made. Thus, preferred are those
analogs where the modifications described in only one or
two of the paragraphs (a)-(e) set forth above in the Dis-
closure section occurs.
Thus, among the preferred analogs of the invention
are those wherein the (7-34), (7-35), (7-36) or (7-37)
form of GLP-1 has been modified only by substitution of a
neutral amino acid, arginine, or a D form of lysine for
lysine at position 26 and/or 34 and/or a neutral amino
acid, lysine, or a D form of arginine for arginine at
position 36 (paragraph (a)). Particularly preferred are
those wherein the amino acid substituted for lysine at
.positions 26 and 34 is selected from the group consisting
of K~', G, S, A, L, I, Q, R, R~ and M, and for arginine at
position 36 is selected from the group of K, K~, G, S, A,
L~ I,~ Q. M. and R~' .
Y ~.~ O C~
.::~ 9vmas~ ' ~crius9noosoo
-25-
Also preferred are analogs wherein the sole
modification is the substitution of an
oxidation-resistant amino acid for tryptophan at position
31 (paragraph (b)). ParticQlarly favored substitutions
are selected from the group consisting of F, V, L, I, A,
and Y.
Also preferred are those analogs wherein the only
modification is at least one of those specific
substitutions set forth in paragraph (c). Particularly
preferred are those analogs wherein combined
substitutions of S.for G at position 22, R at positions
23 and 24 for Q and A respectively, and Q for K at
position 26 have been made, or substitutions of Y for V
at position 16 and K for S at position l8 have been made,, ,
or these substitutions plus D for E at positions 21 have
been made.
Also preferred are analogs wherein the sole modi-
fications are those set forth in paragraph (d). Par-
ticularly preferred among these are those wherein the
2o small neutral amino acid substituted for alanine at posi-
tion 8 is selected from the group consisting of S, S~, G,
C, Cfi, Sar, A~, beta-ala, and Aib;~and/or the.acidic or
neutral amino acid substituted for glutamic at position .9
is selected from the group consisting of Efi, D, D~, Cya,
T, T~, N, N~, Q, Q~, Cit, MSO, and acetyl-K; and/or the
alternative neutral amino acid substituted for glycine at
position l0 is selected from the group consisting of S,
St, Y, Y~, T, T~,_N, N~, Q, Q~, Cit, MSO, acetyl-K, F
and F~; and/or wherein D is substituted for E at position
15.
Also preferred are analogs wherein position 7 alone
has been altered (paragraph (e)). Preferred
substitutions are those wherein the amino acid
substituted for histidine at position 7 is selected from
,;.-::.
WO 91/11457 ' PCT/US91/00500
-26-
the group consisting of Hj~ j~ t j-
Y, Y , F, F , R, R , Orn,
Orn~, M, M~, N-formyl-H, N-formyl-H~, N-acetyl-H,
l
N-acetyl-H~, N-isopropyl-H, N-isopropyl-Hj, N-acetyl-K;
N-acetyl-K~, P, and Pfi.
Also preferred are~embodiments with a combination
of only two of the above-referenced classes of modified
forms, in addition to the follo~ding specific embodiments.
The following specific analogs are preferred:
(H~)7-GLP-1(7-37);
(y)7-GLP-1(7-37);
(N-acetyl-H)7-GLP-1(7-37);
(N-isopropyl-H)7-GLP-1(7-37);
(A~)8-GLP-1(7-37);
(Efi)9-GLP-1(7-37);
(D)9-GLP-1(7-37);
(Dfi)9-GLP-1(7-37);
(Ft)10-GLP-1(7-37)
(S)22(R)23(R)24(Q)26-G~-1(7-37); and
(S)8(Q)9(Y)16(K)18(~)21-GLP-1(7-37).
B. stability Analoas
Preferred forms of analogs with enhanced stability
also have only one, or at most two, amino acid
modifications.
Preferred substitutions for the histidine at
position 7 include the D-forms of acidic or neutral amino
acids or the D-forms of histidines. Preferred are P~,
D~, E~, N~, Q~, L~, V~, I~ and H~.
The histidine at position 7, or a replacement (D or
L), can also be N-alkylated (1-6C) or N-acylated (1-6C).
3p Alkyl groups are straight or branched chain (including
cyclic) hydrocarbyl residues of the indicated member of
Acyl groups are of the formula RCO - wherein R is
alkyl as defined above. Preferred alkyl groups are c-.
propyl, a-propyl and ethyl; preferred acyl are acetyl and
-27-
propionyl. Preferred residues which may be alkylated or
acylated include P, D, E, N, Q, V, L, I, K and H in
either the D or L form.
Preferred substitutions for alanine at position 8
are the D-forms of P, V, L, I and A; also preferred are
the D-forms of D, E, N, Q, K, T, S and H.
It is understood, as is demonstrated below, that
some specific analogs show both enhanced insulin release
stimulating activity and enhanced stability.
Preparation
The analogs of the invention can be prepared using
standard solid-phase techniques for the synthesis of
peptides. As is generally known, peptides of the
requisite length can be prepared using commercially
available equipment and reagents following the
manufacturers' instructions for blocking interfering
groups, protecting the amino acid to be reacted,
coupling, deprotection, and capping of unreacted
residues. Suitable equipment can be obtained, for
example, from Applied BioSystems in Foster City,
California, or Biosearch Corporation in San Raphael,
California.
In a preferred method, the peptides are synthesized
using standard automated solid-phase synthesis protocols
employing t-butoxycarbonyl-alpha-amino acids with
appropriate side-chain protection. Completed peptide is
removed from the solid phase support with simultaneous
side-chain deprotection using the standard hydrogen
fluoride method. Crude peptides are further purified by
semi-preparative reverse phase-HPLC (Vydac C18) using
acetonitrile gradients in 0.1% trifluoroacetic acid
(TFA). The peptides are vacuum dried to remove
acetonitrile and lyophilized from a solution of 0.1% TFA
*Trademark
CA 02073856 2000-02-22
~t~'~~8'~~
.:--...
,::..:..
WO 91/11457 ' PCF/US91/00500
-28-
in water. Purity is verified by analytical RP-HPLC. The
peptides can be lyophilized and then solubilized in
either water or 0.01 M acetic acid at concentrations of
1-2 mg/mL by weight. -
The use of the aforementioned synthetic methods is
needed if nonencoded amino acids or the D forms of amino
acids occur in the peptides. However, for peptides which
are gene-encoded, recourse can also be had to re-
combinant techniques using readily synthesized DNA
sequences in commercially available expression systems.
Formulation and Administration
The analogs of the invention are useful in the
treatment of Type II diabetes. The analogs can be ,
administered systemically in a variety of formulations,
as is generally known in the art. Formulations
appropriate for particular modes of administration for
peptides are set forth in, for example, Reminqton~s
phay aceutical Sciences, latest edition, Mack Publishing
Company, Easton, Pennsylvania. In general, the
formulations utilize an effective amount of the analog or
mixtures of analogs and at least one pharmaceutically
acceptable excipient.
A variety of modes of administration.are effective
in systemic treatment, such as injection, including
intravenous, intramuscular, subcutaneous, and
intraperitoneal injection; transmembrane or transdermal
administration, using suitable suppositories or sprays;
and, if properly formulated, oral administration. Suit-
able excipients for injection include various physiologi-
cal buffers, such as Hank's solution and.Ringer's solu-
tion; suitable transmembrane.or transdermal formulations
contain penetrants such~as bile salts or fusidates; and
typical oral formulations contain protective agents which
~~~j~~n
.aao
.::::T
.:O 91/11457 ' PCT/US91/00500
-29-
inhibit the digestion of the active ingredient. Also
available are various slow-release formulations involving
macromolecular matrices such as pyrrolidones and methyl-
cellulose. Alternate drug delivery systems include
liposomes and microemulsions. A variety of formulations
are workable, and the provision of appropriate formula-
tions for the selected peptides arid administration routes
is generally understood by practitioners.
A typical dosage range for the compounds of the
invention is about l pg/kg-1 mg/kg body weight, although
these are approximations depending upon a large number of
factors including the potency of the analog, its
circulating half-life, the individual characteristics of
the subject, and the like. Optimization of
administration of insulin for diabetic treatment of
individuals is well established, and similar optimization
protocols are employed here:
Examples
The following examples are intended to illustrate,
but not to limit, the invention.
Example 1
Enhanced Insulin Stimulation by Analogs of the Invention
As shown in Figure 2, analogs of the invention
having a variety of substituents modifying the native
structure have been prepared. Some of these analogs have
been tested in the adenylate cyclase assay referenced
above, with the results shown in Table 1.
35
.
w ~~
WO 91/11457 ' PCT/US91/00500
-30-
TABLE 1
ED50 nM
Positive Controls Duplicate AssaSrs
GLP-1(7-37) 0.16 0.25
GLP-1(7-36)(amide) 0.16 0.20
Related Peptides
Glucagon . 80.0 140
Secretin NR -
G~P '10.0 37.5
GRF ~ -
Negative Controls
GLP-1(1-37) >1000 2900
GLP-1(2-37) - -
GLP-1(3-37) 70 81
GLP-1(4-37) 130 ' 200
G~-1(5-37) 150 750-970
Analogs
(H~)~-GLP-1(7-37) l.1 2.2
(Y)7-GLP-1(7-37) 5.0 5.0
(N-acetyl-H)7-GLP-1(7-37) 15.5
(N-isopropyl-H)7-GLP-1(7-37) 15.5
(K)7-GLP-1(7-37) 350.0 - _
(Aj'),8-GLP-1(7-37) 0.40 0.55
(E~')9-GLP-1(7-37) 55.0 74.0
(D)9-GLP-1(7-37) 0.17 0.28
(Dt)9-GLP-1(7-37) 0.90 0.90
(F~)10-GLP-1(7-37) . 12.0 23.0
22
23
24
26
(R) 0.94 1.8
(R)
(Q)
(S)
-GLP-1(7-37)
(S)8(Q)9(Y)16.(K)18(D)21_GLP-1(7-37) 0.31
~~~~~J~..
::0 91/11457 ' PCT/US91/00500
-31-
The various analogs of the invention thus show a
useful range of potencies in the insulinotropic assay.
Exanple 2
~ Enhanced Stability of GLP-1 Analogs
A. Demonstrat:~c ~, of Mode of Inactivation
The GLP-1 (7-37) truncated hormone Was
radioiodinated and the purified peptide was incubated
with plasma and assayed by radiolabel sequencing as
described hereinabove. The sequencing was done on
samples at time zero, 15 minutes and 60 minutes. At time
zero, a single peak of radioactivity was found at cycle
13 indicating no degradation. After 15 minutes, the
amount of radioactivity in cycle 13 was reduced, and that
in cycle 11 was enhanced. After 60 minutes of
incubation, virtually all of the counts appeared at cycle
1l.
It thus appears that a single dipeptidyl
aminopeptidase cleavage is responsible for the
degradation of the GLP-1 (7-37) peptide.
The foregoing results are consistent with
degradation as measured by RIA using N-terminal specific
and C-terminal specific antisera. When incubated with
plasma as described above and tested by RIA, no
w diminution in the ability of the recovered fragment to
inhibit binding of radiolabeled GLP-1 (7-37) to carboxy
terminal-specific antibody was found; however, the
ability to inhibit binding to the amino terminal-specific
antibody decreased almost to zero after 1 hour.
B. GLP-1 (7 -371 Analog's Tested by Radiolabel Seauencina
The radiolabel sequencing method of degradation
analysis was conducted using a GLP-1 (7-37) analog which
35.
.~ e9 i7 i l~ ; . .
WO 91f11457 PGTlUS91/00500
-32-
contained either D-Asp in the 9-position or D-Ala in the
8-position. The results of this assay are shown in
Figure 3. Figure 3A shows the results for (D~)9-GLP-1
(7-3?) and Figure 3B shows the results for (A~)8-GLP-1
(7-37). As shown in these figures, the (Dfi)9 analog
degrades in a manner similar to GLP-1 (7-37); on the
other hand, the (At)g analog showed almost no degradation
after 60 minutes.
C. Analogs Tested by RIA
The N-terminal specific antibody can be used to
measure the degradation of analogs only if it is able to
cross-react with these analogs, which themselves contain
alterations in the N-tenainus. Figure 4 shows the
results for analogs modified at positions 7, 8 and 9.
(Y)7, (Ht)7 and (At)8. appear to be capable, although at
high concentrations, of cross-reactivity; (D~)9 is not:
The cross-reacting peptides were incubated with plasma
for 60 minutes at high concentrations (10-100 nM) and
tested by RIA using RIA against the N-terminal specific
antibody. Consistent with the results in paragraph B,
the (A~)8 analog was not degraded after f0 minutes, nor
was the (Ht)7 analog. However, the (Y)7 analog was
degraded.
D. Analogs Shown Protease Resistant by HPLC
The resistance of various analogs to degradation as
compared to GLP-1 (7-37) was also tested by HPLC as
described above. The incubation in plasma was for 60
minutes; either degradation was not observed or was
complete after this time. The results are shown in Table
~.~'r~ 3 J ;~
.~ J 91/11457 ' PCT/US91/00500
-33-
TABLE 2
Resistance to
Analoa _ Dearadation
(H~)7 GLP-1 (7-37) +
(N-acetyl-H)7 GLP-1 (7-37) +
(N-isopropyl-H)7 GLP-1 (7-37) ' +
(Y)7 GLP-1 (7-37) -
(K)7 GLP-1 (7-37) _
(N-acetyl-K)7 GLP-1 (7-37) t
(S)8(Q)9(Y)16(K)18(D)21 GLP-1 (7-37) -
(A~)8 GLP-1 (7-37) +
(D~)9 GLP-1 (7-37) -
(Et)9 GLP-1 (7-37) -
(Q)9 GLP-1 (7-37) -
25
35
